Assessing the Biocompatibility and Regeneration of Electrospun-Nanofiber Composite Tracheal Grafts

Abstract

Objective: Composite tracheal grafts (CTG) combining decellularized scaffolds with external biomaterial support have been shown to support host-derived neotissue formation. In this study, we examine the biocompatibility, graft epithelialization, vascularization, and patency of three prototype CTG using a mouse microsurgical model.

Study design: Tracheal replacement, regenerative medicine, biocompatible airway splints, animal model.

Method: CTG electrospun splints made by combining partially decellularized tracheal grafts (PDTG) with polyglycolic acid (PGA), poly(lactide-co-ε-caprolactone) (PLCL), or PLCL/PGA were orthotopically implanted in mice (N = 10/group). Tracheas were explanted two weeks post-implantation. Micro-Computed Tomography was conducted to assess for graft patency, and histological analysis was used to assess for epithelialization and neovascularization.

Result: Most animals (greater than 80%) survived until the planned endpoint and did not exhibit respiratory symptoms. MicroCT confirmed the preservation of graft patency. Grossly, the PDTG component of CTG remained intact. Examining the electrospun component of CTG, PGA degraded significantly, while PLCL+PDTG and PLCL/PGA + PDTG maintained their structure. Microvasculature was observed across the surface of CTG and infiltrating the pores. There were no signs of excessive cellular infiltration or encapsulation. Graft microvasculature and epithelium appear similar in all groups, suggesting that CTG did not hinder endothelialization and epithelialization.

Conclusion: We found that all electrospun nanofiber CTGs are biocompatible and did not affect graft patency, endothelialization and epithelialization. Future directions will explore methods to accelerate graft regeneration of CTG.

Level of evidence: N/A Laryngoscope, 134:1155-1162, 2024.

Keywords: Tracheal replacement; animal model; biocompatible splint; regenerative medicine.

Figure 1.. CTG creation procedure.

A. Creation of biomaterial splint. B. PDTG and splint implantation to create CTG.

Figure 2.. Material in vitro degradation test.

A. Percent mass lost in each biomaterial splint composition based on in vitro degradation. B. SEM imaging of in vitro degradation.

Figure 3.. A Gross images at CTG implantation and explantation.

B. Representative MicroCT images of sagittal view at day 14.

Figure 4.

Masson’s Trichrome staining of overall collagen in PDTG (A), PDTG+PGA (B), PDTG+PLCL (C), and PDTG+PGA/PLCL (D). * denotes the splint at day 14; ↕ denotes the patent lumen.

Figure 5.. Macrophage characterization.

A. Representative images of CD68 staining of submucosal macrophage infiltration in different CTG groups. Scale bar= 100 μm. B. CD68+ macrophage quantification in cell number/mm² in submucosa.

Figure 6.. Ciliated epithelial cell characterization.

A. ACT stain for ciliated epithelium and FOXJ1 stain for nuclear marker of functional epithelium in different CTG groups. B. Percentage of ACT coverage on graft epithelium in different CTG groups. C. Number of FOXJ1+ cells present in graft epithelium in different CTG groups.

Figure 7.. Basal cell characterization.

A. K5K14 stained basal cells demonstrating activation. B. Number of K5+ basal cells per mm of graft epithelium in different CTG groups. C. K5+K14+ basal cells to K5+ basal cells in different CTG groups.

Figure 8.. CD31+ endothelial cell characterization in submucosa.

A. Representative images of CD31 stained endothelial cells in different CTG groups. B. Number of CD31+ cells per high powered field in different CTG groups.